In medical inspection, management on safety and sanitation of food, monitoring for environmental conservation, and so on, extraction of a target substance from a sample containing a variety of foreign substances is required for detection and reaction of the target substance. For example, in medical inspection, it is necessary to detect, identify and quantitatively determine a nucleic acid, a protein, a sugar, a lipid, a bacterium, a virus, a radioactive substance or the like which is contained in a biological sample separated and acquired from an animal or a plant, such as blood, serum, cells, urine or feces. In these inspections, it may be required to separate and purify a target substance for eliminating adverse effects such as a background ascribable to foreign substances.
For example, in gene examination, a nucleic acid in a biological sample is amplified by a gene amplification method such as PCR (polymerase chain reaction), and a sequence of the nucleic acid is determined. For efficiently amplifying a nucleic acid by a gene amplification method such as PCR, it is necessary that the nucleic acid be extracted and purified from a biological sample containing various foreign substances, and provided for the reaction. In extraction and purification of a nucleic acid in a biological sample, a phenol/chloroform method has been normally used for a long time. However, the phenol/chloroform method has the problem that the operation is complicated, reagents to be used are harmful, and much cost is required for treating a waste liquid.
Patent Document 1 proposes a method for extracting and purifying a nucleic acid from a biological sample by particle manipulation (Boom method), utilizing the nature of a nucleic acid being specifically adsorbed to silica. In this method, first, a solution obtained by treating a sample in the presence of a protease and a surfactant is mixed with silica particles in the presence of a chaotropic substance such as a guanidine salt, an iodide salt or urea to adsorb a nucleic acid to the surfaces of the particles. The silica particles to which the nucleic acid is adsorbed are washed several times for removing foreign substances. Thereafter, the silica particle composite is added in a solution (eluting liquid) having a low salt concentration, so that the purified nucleic acid is eluted in the eluting liquid to obtain a nucleic acid purified sample. Chemical operations applying such particle manipulation are not only targeted at a nucleic acid, but also increasingly applied to, for example, an immunoassay using an antigen-antibody reaction (see, for example, Patent Document 2).
In operations using particles, it is necessary to properly separate particles (solid phase) to which a target sample such as a nucleic acid is adsorbed and a water-based liquid (liquid phase) containing foreign substances which are not adsorbed to the particles. As such a separation operation (B/F separation), a method has been proposed in which a water-based liquid with particles dispersed therein is made to exist as droplets in a liquid immiscible with the water-based liquid (e.g., oil), a nucleic acid or the like is adsorbed to the surfaces of the particles in the water-based liquid droplets, and only particles to which the nucleic acid is adsorbed are then moved into the oil. In addition, a method has been proposed in which particles are moved by magnetic field manipulation using silica-coated magnetic particles or the like for facilitating selective movement of particles.
When a water-based liquid exists as droplets in an oil, the fluid resistance of the oil, the water-repelling force at the interface between the oil and the water-based liquid, the interaction between hydrophilic groups on the surfaces of particles and the water-based liquid, and the like can act as a force for retaining the position and shape of the droplets. Accordingly, when only magnetic particles are to be moved and separated from water-based liquid droplets into an oil, the droplets follow the magnetic field along with the magnetic particles, and therefore it is not easy to separate only particles from the water-based liquid droplets into the oil.
In view of this problem, a method for selectively moving only particles into an oil by suppressing movement of water-based liquid droplets has been proposed. For example, Patent Document 3 discloses a method in which the inner surface of a container, in which droplets and an oil are enclosed, is provided with a physical structure such as a partition wall to block the droplets, and only magnetic particles are moved and separated from a part of a gap. However, in the method disclosed in Patent Document 3, it is necessary to subject the oil enclosing portion to special microprocessing, and therefore the configuration of a device is complicated. Even when a physical structure is provided, the water-based liquid deposited on the peripheries of particles cannot be completely blocked, and therefore B/F separability may not be sufficient.
Patent Document 4 discloses a method in which by applying an electric field to the carrying surfaces of droplets, only magnetic particles are separated with the droplets immobilized at a predetermined position on the carrying surfaces. However, this method has the problem that since droplets cannot be immobilized unless an electric field is constantly applied to a device, the configuration of the device is complicated, and moreover, the method cannot be used in an environment which is short of supply of energy such as electric power. Therefore, the method is not suitable for use in an environment where infrastructure is not fully developed like a developing country or the like, and in an environment where it is required to examine a large number of specimens in a short time, for example, in a state of emergency such as terrorism or disaster. The device to be used in manipulation of a biological sample is preferably disposable for suppression of a problem such as contamination, prevention of infection, and so on. From the above-mentioned viewpoint, it is required to develop a chemical operation method and a device to be used therein that is capable of performing purification, examination and the like conveniently, quickly, at a low cost and in every site.
Patent Document 5 discloses a method in which a low-molecular gelling agent such as a hydroxy fatty acid, a dextrin fatty acid ester or a glycerin fatty acid ester is added to a liquid (silicone oil or the like) immiscible with a water-based liquid to form a physical gel, so that water-based liquid droplets are immobilized at a predetermined position in the oil. The physical gel turns into a sol having fluidity at a temperature higher than the sol-gel transition temperature, and turns into a semi-solid gel having no fluidity at a temperature lower than the sol-gel transition temperature. Accordingly, when the water-based liquid is added in a sol-like oil heated to a temperature equal to or higher than the sol-gel transition temperature, and the oil is then gelled by cooling, water-based liquid droplets are embedded and immobilized in the gel. According to this method, only magnetic particles can be moved into a gel by magnetic field manipulation while water-based liquid droplets are immobilized at a predetermined position in the gel. Accordingly, a water-based liquid containing foreign substances and particles with a target sample immobilized thereon can be conveniently and efficiently separated.
In the method of Patent Document 5, when the oil is heated and thereby solated, the droplets can be released from the embedded and immobilized state to move in the sol-like oil. Thus, the inside of a container, in which the physical gel is enclosed, is given a temperature gradient, so that a region where the oil is in a gel state and a region where the oil is in a sol state coexist, a temperature cycle of PCR can be carried out by performing nucleic acid extraction and purification operations in the low-temperature gel region, and moving particles along with droplets in the oil in the high-temperature sol region. According to this method, extraction and purification of a nucleic acid requiring immobilization of droplets at a predetermined position, and PCR requiring movement of droplets can be performed in one device while a hermetically sealed state is maintained.